The Anion-Exchange Separation of Ribonucleotides - Journal of the

Norman E. Hoffman and John C. Liao. Analytical Chemistry ... Calvin S. McLaughlin and Vernon M. Ingram ... Burton E. Tropp , John H. Law , and John M...
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April, 1950

ANION-EXCHANGE SEPARATION OF RIBONUCLEOTIDES

rigorous examination may establish this application of the techniques described. Presumably the nucleotides and nucleosides of thymus nucleic acid may be analyzed by these procedures; however, adequate standards are not yet available to test this application. Summary Solvent systems for paper chromatography of purine and pyrimidine derivatives of yeast nucleic acid are described and the use of the method for analysis of complex mixtures of these compounds by two-dimensional chromatography reported.

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Location of compounds on the paper chromatogram is achieved by ultraviolet fluorescence. It has been found that the adenylic acid derived from yeast nucleic acid is resolved by chromatographic procedures into two components which may be chemically and enzymatically degraded to adenine and adenosine. It is therefore suggested that the two adenylic acids differ with respect to the location of the phosphoric acid moiety on the ribose chain and are adenosine-2phosphoric acid and adenosine-3-phosphoric acid, respectively. RECEIVED MARCH3, 1949

BIOLOGYDIVISION OF OAK RIDGEN A T I O S A L

LABORATORY’]

The Anion-Exchange Skparation of Ribonucleotides BY WALDOE. COHN Investigations of nucleic acid chemistry, constitution and metabolism, particularly where tracers are employed, depend to a great degree upon the development of methods for the quantitative separation of the various chemical and enzymatic degradation products of the larger molecules. For greatest usefulness, these methods should be operable a t the lowest detectable levels of concentration and amount of material and they should give adequate resolution-complete, if possible-of closely related substances. The classical methodsla are based principally upon precipitation which, because of solubility limitations and coprecipitation, do not, in general, meet such quantitative demands as well as do those methods based upon solvent extraction or ion-exchange, which have found such utility in the isolation of carrier-free radioisotopes. Previous work in the development of ion-exchange procedures for the quantitative separation of fission product ions, including the rare earth elements, from their mixtures a t trace concentrat i o n ~led ~ ,to~ the ~ ~ choice of ion-exchange as a likely mechanism to exploit in order to develop better separation methods for the nucleotides, nucleosides and free bases. The fact that nucleotides and their degradation products contain both acid and basic groups offered the possibility of using both anion and cation exchangers while the differences in pK from compound to compoundla (1) Operated by Carbide and Carbon Chemicals Corporation under Contract No. W-7405-Eng-26 for the Atomic Energy Commission, Oak Ridge, Tennessee. (la) P. A. Levene and L. W . Bass, “Nucleic Acids,” Chemical Catalog Co., Inc., New York, N. Y., 1931. (2) W. E. Cohn, in “Advances in Biological and Medical Physics,” Vol. I, Academic Press, New York, N. Y., 1949; also, N Q U QMed. ~ Bull. S u p p l . , Mar.-April, 1948, p. 42. (3) E. R. Tompkins, J . X. Khym and W. E. Cohn, THIS JOURNAL, 69, 2769 (1947). (4) W. E. Cohn, G. W. Parker and E. R. Tompkins, Nucleonics, 3 , No. 5 , 22 (1948). ( 5 ) W.E . Cohn and H. W .Kohn, THISJ O U R N A L , 70, 1986 (1948).

indicated that these might prove as conclusive in effecting organic separations as are the complex dissociation constants and base strengths in the rare earth separations. The experiments and results described below (and described in preliminary form earlier) 6,7 indicate that ion-exchange is a separation tool of the required accuracy and sensitivity and, as with any quantitative and sensitive separation method, it can serve also as a means of analysis, investigation and production. Preliminary Considerations.-To effect a n ion-exchange separation of the members of a family of ions, i t is necessary to find a set of conditions under which each exhibits a different degree of affinity (distribution coefficient) with respect to the exchanger. While this affinity is governed by a number of variable^,^*^ i t is reasonable to assume that, in a group as closely related as the monoribonucleotides, the net charge per ion will be the most important one. Since these substances possess both acid (phosphate) and basic (amino) groups, the p H of the medium determines the net charge by determining the degree of dissociation of these groups. Hence, a preliminary calculation was made of the degrees of dissociation of these groups as a function of p H , using the PK values quoted by Levenela; these calculations are presented graphically in Fig. 1. It is immediately apparent from Fig. 1 that (1) the order of increasing net negative charge per molecule is cytidylic, adenylic, guanylic and uridylic acid; ( 2 ) cytidylic and adenylic exhibit a net positive charge, and hence are cations, a t p H values below about 2.5 and guanylic below 1.5, while uridylic remains anionic down to p H 0, which facts are consistent with the cation-exchange sep(6) (7) (8) (9)

W. E. Cohn, Science, 109, 377 (1949). W. E. Cohn, THISJOURNAL, ‘71, 2276 (1949). E. R. Tompkins. J . Chem. Educ., 96, 32, 92 (1949). R. Kunin. A d . Chcm., 21, 87 (1949).

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with acid and base before being converted to the salt form desired for a particular experiment. .Columns were used repeatedly without change of resin, often without backwash or resuspension, until accident or change in type of experiment warranted discarding them. One has been used in thirty separations without noticeable loss in combining capacity or efficiency. Test Substances.-As a source of mixed monoribonucleotides, barium hydroxide hydrolysates of yeast nucleic acid were u ~ e d . ~ ~ ~Commercial 14 adenylic, guanylic and cytidylic acids were employed without purification as the impurities lent themselves to the investigations in hand. Solutions of uridylic acid were prepared by cation-exchange methods,B identified by spectral properties" and standardized by phosphorus analysis .ll Procedure.-The columns were prepared for use by washing with solutions of stronger eluting power than any which were to be used in the experiment (e. g., 1 N hydrochloric acid if replacement by C1- was contemplated) until influent and effluent were identical in PH and optical density a t 240-280 mp. Following a water wash, the test substance, in amount usually less than 5% of the exchanger capacity, was adsorbed from a solution of low eluting power (e. g., pH 8, anion concentration $0.01M). The column was again washed with water and elution with the desired reagent was commenced. Zero volume was taken as the point when the effluent attained the same PH and ionic strength as the influent solution.

+0.4

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-0.4

2 -0.6

- 0.8

- 1.0 3 2 1 0 PH . Fig. 1.-Net charge per molecule of ribonucleotide as a function of pH (calculated from data of Levene). 6

5

4

arations reported previously6; (3) a t PH values above 5, all are strongly negative. From these observations, i t would seem simplest to adsorb the mixture at PH values of 6 or greater, and to remove the nucleotides in the order cytidylic, adenylic, guanylic and uridylic acid by lowering the PH in a stepwise fashion, increasing the anion concentration with salt near the end if necessary to avoid strongly acid conditions which might hydrolyze the compounds. This was the plan of the experiments reported below.

Results Although many conditions, varying from pH 1.8 to 5.6 with various anions and varying anion concentrations, were studied, i t is necessary to present only a few typical experiments a t the various extremes before summarizing the data obtained. A rapid partial separation, utilizing a stepwise pH procedure as outlined above, is shown in Fig. 2 . The spectrophotometric ratios (i. e., ratios of optical densities a t two wave lengths) of the fractions collected indicate a tendency for guanylic acid to follow uridylic acid, the reverse of the predicted order. The degree of sep-

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Experimental Apparatus.-The type of column is described by Tompkins.am* An automatic sample changer was used to change receivers a t fixed time intervals, thus permitting extended runs t o be made conveniently. A Beckmann ultraviolet spectrophotometer was employed to assay and identify the samples, taking advantage of the characteristic ultraviolet spectra of the several substances being investigated. The dependency of the shape of these spectra upon pH offers a further method of identification. Paper chromatography also served as a means of identification. l0,11 Ion-exchangers:-The experiments utilized principally 200-400 mesh Dowex-1,12 a strong-base anion exchanger; Dowex 212 and Amberlite IRA-4001a were also employed with similar results. The exchangers were washed free of fines by decantation, slurried into the columns and washed (10) We are indebted to Dr. C. E. Carter for chromatographic and other analyses" and also for the nucleic acid hydrolysates and other preparations used in these experiments. (11) C. E. Carter, THISJOWRXAL, 72, 1466 (1950). (12) Obtained through the courtesy of Dr. L. A. Matheson, D o w Chemical Co., Midland, Michigan. (13) Resinous Products and Chemicals Co., Washington Square, Philadelphia, Pa.

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50 100 150 M1. through column. Fig. 2.-Rapid separation of nucleotides : exchanger, Dowex-1, 0.9 sq. cm. X 2 cm.; solution, hydrochloric acid a t 0.8 ml./min.; test material, -4 mg. of mixed nucleotides. (14) H. S. Loring, P. M. Roll and J. G. Pierce, J. Bid. Chcm., 174, 729 (1948). (15) J. M. Ploeser and H. S. Loring, ibid., 178, 431 (1949).

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ANION-EXCHANGE S E P A R A T I O N OF RIBONUCLEOTIDES

aration of cytidylic and adenylic acids is apparent. Free bases (adenine, guanine, uracil, cytosine) or ribosides exhibit practically no affinity for anionexchangers a t PH values below 8 and hence are removed, if adsorbed a t all, ahead of cytidylic acid.6 As predicted, separations a t PH values above 4 are less satisfactory and i t is necessary to use longer columns in order to achieve even a separation into pairs of nucleotides, as shown in Figs. 3 and 4. In addition, the affinityof the nucleotides

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poses of concentration (see below). Nevertheless, such a procedure is satisfactory for simple analysis as the spectrophotometric ratios are suffcientiy different to permit calculation of the composition of each of the two fractions (pyrimidine nucleotides and purine nucleotides). In addition, satisfactory resolution of the isomers of adenylic and of inosinic acids can be obtained under these same conditions; this, as well as the proof that two kinds of adenylic acid exist in hydrolysates of yeast nucleic acid, is discussed elsewhere.ll9l6

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100 200 300 M1. through column. E F i g . 3.-Rapid separation of mononucleotides: exchanger, Dowex-1 (Cl-), 0.74 sq. cm. X 12 cm.; solution, 0.1 M sodium chloride in 0.01 M acetate buffer, pH 5.6, 0.7 ml./min.; test material, -4 mg. of purified nucleotides.

200 4000 200 400 600 800 M1. through column.

.Fig. 5.-Separation of nucleotides: exchanger, Dowex-1 (formate), 0.74 sq. cm. X 13 cm.; solution, formic acid, 0.7 ml./min.; test material, 3 mg. of mixed nucleotides from barium hydroxide hydrolyzate of yeast nucleic acid.

Longer columns and higher distribution coefficients give better separations, as demonstrated in Fig. 5. Utilizing formic acid in place of hydrochloric (giving better pH control, 0 more available anion and a lower speN" 1.0 cific replacement power than C1- on Dowex-l)," adenylic acid is comh 0.8 U .* pletely separated from the other nucleotides and divided into two parts. 4 0.6 (This experiment is the first in which U practically complete resolution by ion-exchange of the two adenylic acid 0.4 isomers derived from yeast nucleic 0.2 acid was obtained; this separation was verified by paper chromatography and by many subsequent separations and tests.11-16It should also be noted 0 1.0 2.0 3.0 4.0 that uridylic now follows guanylic, as Liters through column. Fig. 4.--Separation of mixed monoribonucleotides: exchanger, Dowex-1 originally predicted, contrary to the (Cl-), 10 cm. X 0.94 sq. cm.; solution, 0.02 M sodium chloride in 0.01 M calculations of Fig. 2 (and of Fig. 6, acetate buffer, PH 5.6, 1.4 ml./min.; test material, -40 mg. of mixed below) 1. The 'best separations of the nucleonucleotides from barium hydroxide hydrolyzate of yeast nucleic acid. tides of ribonucleic acid with hydrofor the exchanger here is so much greater than a t chloric acid have been obtained a t pH values p H 2-3 that the anion concentration must be in(16) C. E. Carter and W. E. Cohn, to be published; Federation creased considerably to achieve comparable elu- Proc,, 8, 190 (1949), tion rates, thus hindering readsorption for pur(17) L. A. Matheson, personal communication. 1.4

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been mentioned above in connection with Figs. 2,4, 5 and ,Barer and Riboridre 6. I n Fig. 7, a separation of 0.6 a mixture of several free bases and nucleosides from adenylic acid, representing the nucleotides, is shown to prove this 0 point. I n this experiment, CD c.1 the concentration of anion v d 0.4 (formate) was kept constant throughout, the @H being shifted by changing the proQuanylic Acids Adaylic Acids portions of ammonium hyo b .-v9 o b droxide and formic acid in the solution. Satisfactory 0" 0.2 resolution of the pairs shown can be attained by more careful adjustment of conditions; this has already been demonstrated for the five bases6and 0.0 for cytidine and uridine.18f19 0.5 1.0 1.5 0.5 1.0 1.5 2.0 2.5 3.0 (The probable positions of Volume of eluting solution, in liters. the latter two substances, Fig. 6.-Separation of mononucleotides of rat liver ribonucleic acid by anion- obtained from experiments, exchange: exchanger, Dowex-1, 12.5 cm. X 0.74 sq. cm.; eluent, dilute hydro- conducted by Mr. J. X. chloric acid, 0.5 ml./min.; test material, ~ 1 mg. 4 of mixed nucleotides from Khym in this Laboratory, are shown in brackets in Fig. 7, barium hydroxide hydrolysis of rat liver nucleic acid in 10 ml. of water. ,Slat 0,002 I HCI

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in the neighborhood of 2.7. 1 A separation of a mixture of nucleotides obtained from a barium hydroxide hydrolysate of ribonucleic acid from r a t liver a t @H2.7 is shown in Fig. 6. The two adenylic acid peaks are ,clearly shown while uridylic now precedes guanylic. Of particular interest is the observation of two peaks in the latter. The eluate was therefore divided into the three fractions (a, b, b) shown in Fig. 6. a When these three fractions were chromatographed,lOjll the samples a and b migrated to two slightly different posib tions, while sample a occupied both positions. All three fractions were de0 200 600 0 700 0 200 409 693 877 0 339 0 233 433 673 graded to manine by acid,1° M1. through column. which is taken evidence Fig. 7.-Separation of bases, ribosides and adenylic acid: exchanger, Dowex-1, 13 that both peaks are due to cm. X 0.74 sq. cm.; rate, 0.5 ml./min. guanylic acid. More evidence is necessary in order to establish the possi- although they were not included in this particular ble existence of two guanylic acids, analogous to run.) Thus, i t is possible to separate most of the the two adenylic acids.11316 The nature of the free bases, nucleosides and nucleotides of ribonusmall peak between cytidylic and adenylic acid is cleic acid from one another in one column run; not known; in amount, i t was too small to be this procedure can, therefore, be used for the quantitative analysis of acid hydrolysates of nuanalyzed. The removal of free base and ribosides ahead of (18) T. Natuuc, 161, g31 (1948), (19) R.J. c . Harris and J. F. Thomas, i b i d . , 161, 931 (1948). (i. e., a t higher @Hvalues than) cytidylic acid has

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April, 1950

ANION-EXCHANGE SEPARATION OF RIBONUCLEOTIDES

cleic acid, which yield adenine, guanine, cytidylic and uridylic acid,20in place of the laborious and less accurate precipitation methods, as well as for other types of hydrolysates. Attempts to obtain complete separations in pH regions above 3 have not been successful as certain peaks are too close together. A complete summary of the data, covering the entire range from pH 1.8 to 5.6, is given by the points in Fig. 8. The points and the broken lines connecting them denote the positions of the several peaks, these positions being proportional to the distribution coefficients,21 with respect to that of adenylic acid a. When used in conjunction with the absolute data on adenylic acid a given in Table I, the curves in Fig. 8 make i t possible to estimate the TABLE I ML. TO PEAKOF ADENOSINE;PHOSPHATE (ADENYLIC ACIDa ) FOR DOWEX-1COLUMN, APPROXIMATELY 0.74 SQ. CM. x 13.5 CM. (10 ML.) Solution

Formic acid

HC1

0.01 dlacetate buffer PlUS r\’dC1

Concentration (MI

gH

hI1. to peak

1 0.25 .1 ,005 ,004 .002 .0016 .01 .01 .02 .02 .10

1.8 2.2 2.4 2.3 2.4 2.7 2.8 3.6 4.6 4.6 5.6 5.6

25 100 340 70 110 ROO 900 800 1800 800 2100 150

positions of the peaks of all ribonucleotides for a given column. The solid curves in Fig. 8 express the calculated net charge of the same nucleotides, again relative to adenylic acid, using the data summarized in Fig. 1; the curves for cytidylic and uridylic acid have, however, been normalized by the arbitrary factor 0.37, the significance of which remains to be determined. While use of this factor gives a good coincidence of experimental and calculated values for uridylic acid, the fit for cytidylic is less satisfactory. This may be due to the fact that cytidylic acid is always eluted first, a t low volumes, with a corresponding large uncertainty in zero point (pH equilibrium) and hence position of peak. The data in Fig. 8 indicate clearly the reversal of the relative positions of uridylic and guanylic acid which takes place a t PH 2.0. Indeed, i t is apparent that uridylic can be eluted simultaneously with guanylic acid a t fiH 2 , with adenylic acids a t pH 3.3-3.5, or with cytidylic acid a t p H values above 4. Also apparent is the fact that the optimum separation of all nucleotides a t a single $H occurs a t about 2.9 (dotted line). ( 2 0 ) G. Schmidt, R. Cubiles and S. J. Thannhauser, Cold. S p y . H b v . Sym. X I I , 161 (1947). (21) E. R. Tompkins and S. W. Mayer, THISJ O U R N A L , 69, 2859 (1947).

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2

5:

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4 4

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0.1 0.3 0.6 1.0 3 6 10 30 Fig. &-Observed relative distribution coefficients (points, broken lines) and calculated relative negative charges (solid lines) of nucleotides as functions of PH.

Although the data presented on separations in the region of PH 2.9 (e. g., Figs. 6 and 7) indicate that the process may be a slow one and, therefore, somewhat impractical, i t should be pointed out that the volume (and hence time) required to remove a given substance from the column a t a fixed pH is dependent upon the anion concentration of the solution. While this point has not been thoroughly investigated, i t appears, as might be expected, that the absolute peak distances (distribution coefficients) are approximately proportional to the (monovalent) anion concentration in the p H range of 2 to 5 where the nucleotides are monovalent anions. Therefore, the time of separation of a mixture into its component nucleotides may be reduced several-fold by increasing the anion concentration after the elution of each substance. Thus, each can be obtained in a small volume, comparable to those of cytidylic and uridylic acids in Fig. 6. Alternatively, as has been shown previously7 and also in Figs. 2,5 and 6, the PH can be changed a t appropriate intervals. The use of formic (or acetic) acids for elution in the p H 2-3 region has three advantages over dilute hydrochloric acid: (1) PH regulation is more exact; ( 2 ) the neutralization of the nucleotides, usually absorbed as divalent anions, is more rapidly achieved, eliminating large dead volumes; (3) simple evaporation may be used to recover the products. However, dilute hydrochloric acid has one very marked advantage in that subsequent concentration by ion-exchange, which requires the conversion of the effluent to a solution of PH 8-10 with low (< 0.01 M ) anion concentration, is possible by the simple addition of a small amount of ammonium hydroxide. Such a solution can readily be stripped of its nucleotide content on 10-fold smaller columns, from which the product can be recovered in concentrated (1-10 mg./ml.) solution by elution with 0.02-0.1 M hydrochloric acid. In practice, it has been possible practically to saturate a 2 ml. column with adenylic acid (about 150 mg.) from 2 liters of 0.01 M C1- solution a t $H 9; subsequent elution, with 0.1 M hydrochloric acid, yields a solution of 5-10 mg./ml., from which the adenylic acid crystallizes on standing. This

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manufacture of both. Of considerable importance in any of these applications is the fact that the shape of the elution curve, within the range of con-' ditions investigated, is that of the normal curve of error, as predicted by the equilibrium theory of Mayer and Tompkins.22 It is, therefore, possible to utilize the mathematical relationships of the normal curve of error to predict quantitatively the conditions necessary for a given degree of separation of two substances (length of column, the ratio of the distribution coefficients), the recoveries a t given volumes of effluent solution, the concentration a t the maximum relative to the distribution coefficient (position of the peak), etc., as has already been demonstrated in the rare earth separations.22 (A full exposi0 1.0 2.0 3.0 4.0 5.0 tion of these applications is Liters HCl (from PH adjustment). given by Tompkins.8) Fig. 9.-Separation of mixed ribonucleotides: exchanger, Dowex-2 (Cl-), 0.94 sq. - The approach of elution cm. X 12.5 cm.; solution, 0.003 M hydrochloric acid, 0.8 ml./min. curves for the nucleotides to the theoretical is demonstrated recycling procedure has been used to prepare in Fig. 9, in which theoretical curves are supergram amounts of the two adenylic acid isomers, imposed upon the elution curves of uridylic and in crystalline form, no other operations being guanylic acids obtained in a single run a t constant necessary. Coupled with a recording flow spec- PH. The theoretical curves were calculated from trophotometer and a recording pH meter, this the formula22 procedure, utilizing a 33 sq. cm. column, has 1 = d F ( F / C - 1) (1) afforded a simple method for isolating pure nucleotides on a gram scale; this will be described in where F and C are the effluent volumes to a given detail elsewhere. point and to the maximum of the elution curve, No attempt has been made to determine opti- respectively, while the constant p (number of mum flow rates. Rates up to 1 ml./sq. cm. plates) is obtained from a simple modification of minute yield elution curves that are apparently the Mayer-Tompkins formula2* symmetrical and practically identical with the namely normal curve of error shape predicted by the p = 2?r (CpLm*x,)2, at peak equilibrium theory of Mayer and TompkinsZ2 p = z r ( concentration total solute X ml. t o peak)* ( 2 ) while rates of 2 and 4 do not (cf. Fig. 6 versus the curve given in'). It would seem, then, Since the area under the normal curve of error that flow rates of about 1 ml./sq. cm. min. (at (amount eluted) corresponding to a given value room temperature) permit the attainment of of t (ordinate of the curve of error) is obtainabIe equilibrium between the resin beds employed an rl from probability tables, only three values are the nucleotides. necessary in order to solve, say, for total solute Discussion present: concentration a t the peak, volume to Although these experiments were undertaken peak, and p. Although 9 may vary, on a given for the purposes stated in the introduction, the column, from compound to compound (cf. Fig. 9) results reported here and previouslys have per- and from one eluting condition to another, both haps much greater significance for such basic i t and the volume to the peak are constant for a problems of nucleic acid chemistry as the quanti- given set of conditions and a given compound and tative assay of known constituents, the detection can be determined from a standardization experiand isolation of unknown constituents and the ment. Therefore, to solve for the total solute present in an unknown sample, as in a quantitative (22) S. W. Mayer and E. R. Tompkins, THISJ O U R N A L , 69, 2866 analysis, i t is necessary to determine only the (1Y17).

F:

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concentration a t the peak or to calculate this fit the few points determined by elution seems to value, using (1) and the probability tables, from be about 0.5, which is not too far-considering the the concentration after a known volume ( F ) number and accuracy of the determinationsof effluent has passed through the column. It is from the 0.37 factor noted for the pyrimidine also possible, once p is known, to predict the frac- nucleotides. It should be recalled that the nucleotides were tion eluted when the effluent has arrived a t a given fraction of the peak concentration. Thus, when adsorbed from solutions of alkaline pH, where the elution curve has dropped to 50% of its maxi- all are very soluble, in the topmost layers of the mum, 88% of the solute has been removed; for exchanger bed (as proved by the concentration a 95% recovery, it is only necessary to proceel data mentioned above). The resins employed to 25y0 of the peak concentration; etc. Since have apparent combining capacities of about 2-3 these relationships hold on the ascending portions meq. per gram of exchanger; thus, the concenof the curves as well, their application to deter- tration of nucleotide in the exchanger may be as mine the degree of overlap of two substances in a high as one millimole per gram. The elutions were then carried out at lower PH values, beseparation procedure is obvious.8p22 These useful applications, as well as the use of tween 5.6 and 1.8, and, therefore, amount to a symmetry as a criterion of a single component, process of dissociating a precipitated acid in a are possible only with elution procedures which solution in which i t is more (pyrimidine nucleoresult in theoretical curves. Since these cannot tides) or less (purine nucleotides) soluble. Indeed, i t has been found possible, a t very low be obtained when conditions vary during the course of a separation, i t is advisable, in quanti- pH and high chloride concentration ( e . g., 0.1 tative work or when searching for new substances, N hydrochloric acid), to elute adenylic acid from to keep flow rate, PH, etc., constant throughout the exchanger a t such a rate that it forms a superto avoid the asymmetry which results from saturated solution, from which i t crystallizes in the receiver or, if the flow rate be slow enough, variations in such conditions. Of considerable interest, from a theoretical in the exchanger bed itself. In the latter case standpoint, is the constant divergence of the the crystals may easily be seen dispersed throughobserved relative distribution coefficients of the out the bed. This indicates that the concenpyrimidine nucleotides from the values predicted tration of nucleotide in the resin bed can exceed on the basis of the ionization constants of the greatly its solubility in the aqueous medium, a phosphate and amino groups. The data (pre- necessary condition in the hypothesis that solusented in Fig. 8) indicate that the pyrimidine bility is responsible for the differences in distribunucleotides are three times less strongly adsorbed, tion coefficient noted. These methods are being applied in the manner per unit charge on the molecule, than the purine nucleotides or, conversely, that the latter are indicated to the analysis of the products of the three times more strongly adsorbed than would be enzymatic digestion of nucleic acid, to the projudged from net charge considerations alone. It duction of uridylic acid and of the various nucleowould seem, in view of the similarities in molecular tide isomers discussed, and to the quantitative weight, constitution, valencies and concentrations assay of the nucleotide content of nucleic acids of these ions, that degree of ionization would be from various sources. It is intended to apply the sole determining factor. them in similar fashion to the isolation of desoxyIn searching for an explanation of the observed ribonucleotides and other nucleotide material of effect, two family differences between pyrimidine interest in our investigations, including polynuand purine nucleotides come to mind: the much cleotides. greater solubility of the formerla and the presence Acknowledgment.-It is a pleasure to acknowlof the extra imidazole ring in the latter. Evi- edge the continued advice and assistance of Dr. dence favoring the former over the latter as the C. E. Carter in the caurse of these experiments, cause comes from a few (as yet unpublished) in particular for the chromatographic analyses and observations on inosinic acid, which possesses nucleic acid hydrolysates. the high solubility in water characteristic of the Summary pyrimidine nucleotides but also the imidazole ring of the purine nucleotides. The inosinic The nucleotides of ribonucleic acid have been acidslO corresponding to the 5, a and b adenylic separated from each other and from the correacids are eluted in that orderI6 ahead of the sponding free bases and ribosides by successive orresponding adenylic acids a t PH 5.6 but follow elution from strong base anion exchangers with them, again in order, a t PH 1.8; a t the latter weak acids and buffers of controlled pH and anion pH, they are scarcely separable from guanylic concentration. The order of elution a t pH 2 is and uridylic acids. This behavior relates them to cytidylic, adenylic, guanylic and uridylic acid; a t uridylic acid (which, like them, possesses no amino PH 2.5-3, uridylic precedes guanylic acid; at pH group). The factor necessary to normalize the 5.5, uridylic and cytidylic are removed together theoretical curve for inosinic acid (based upon as are adenylic and guanylic acid. I n all cases, dissociation constants only as shown in Fig. 1) to the bases and ribosides precede cytidylic acid (

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and may be separated from each other as well as from the nucleotides during the procedure. The three isomers of adenylic and of inosinic acids may be separated.

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The applications of this technique to analysis, isolation, investigation and manufacture are indicated. RECEIVED JULY 5, 1949

SCHOOL OF CHEMISTRY, UNIVERSITY

OF

MINNESOTA]

The Preparation of 2-Alkylthioimidazoles BY R. M. DODSON AND FRANK Ross’

It has recently been shown that 2-benzylthio- methyl sulfate yields 1-methyl-4-phenylimidazole 4 ( 5 )-phenylimidazole could be prepared by the and 1-methyl-5-phenylimidazole in the ratio of reaction of S-benzylisothiourea with phenacyl 4.8 to 1. We did not succeed in isolating any from our bromide.2 This reaction has now been extended l-methyl-2-benzylthio-5-phenylimidazole to the preparation of l-substituted-2-alkylthio-4- methylation. By analogous reactions, N-phenyl-S-benzylisophenylimidazoles by the condensation of Nsubstituted-S-alkylisothioureas with phenacyl thiourea (V) and N-phenyl-S-methylisothiourea bromide. Thus, l-methy1-2-benzylthio-4-phenyl-(VII) were condensed with phenacyl bromide imidazole (11) was prepared in low yield from N- to give 1,4-diphenyl-2-benzylthioimidazole(VI) methyl-S-benzylisothiourea (I). Although two and 1,4-diphenyl-2-rnethylthioimidazole(VI 11), isomeric compounds, l-methyl-2-benzylthio-4- respectively, in yields of 51 and 40%. None of was found phenylimidazole (11) and 1-methyl-2-benzylthio- the 1,5-diphenyl-2-alkylthioimidazole 5-phenylimidazole, could be expected from the in either of these preparations. Compounds reaction, only compound I1 was found. The VI and VI11 were cleaved to the same 1,4-distructure of compound I1 was established by its phenyl-2-thiolimidazole (IX) with phosphorus cleavage to l-methyl-2-thiol-4-phenylimidazoleand iodine in glacial acetic acid,4 and the 2-thiol(111) with phosphorus and iodine in glacial acetic imidazole (IX) was then oxidized to 1,4-diphenylacid, followed by oxidation of I11 to the known imidazole (X). In order to establish the structures of com1-methyl-4-phenylimidazole (IV). pounds VI and VIII, 1,4-diphenyl-2NHz + C6H5-C-cH~Br + Rz-S-C/ N-c-c6H5 thiolimidazole (IX) was independently Rz-S-C/ 11 ‘ N C H synthesized from N-phenacylaniline \N II 0 I and potassium thiocyanate. This 1,4I RI RI diphenyl-2-thiolimidazole was then al11, RI = ‘3% R2 = CeH6CH2I, R1 = CHs, Rz = CsH5CHzkylated with benzyl chloride and with V,RI = C6H6, RZ = C ~ H ~ C H Z - VI, RI = C6Hh RZ = C6HsCHZmethyl iodide to produce 1,4-diphenylVIII, R1 = C6H5, Rz = CH3VII, R1 = CeHa, Rz = CH32-benzvlthioimidazole (VI) and 1.4-di1 phenyl--2-methylthioimidazole (VIII) , iespectively. I

/N-c-c6Hj

+

/N-C-CsHa

A comparison of the above reactions of S-alkylisothioureas with the alkylaI I tion of N-substituted benzamidines RI RI indicates that the reactions are similar. IV, R1 = C6H5CHI111, RI R1 = = CHsPyman5has shown that N-phenylbenz1x9 X, R1 amidine monomethylated in good yield l-Methyl-2-benzylthio-4-phenylimidazole (11) on the nitrogen holding the phenyl group. The was independently synthesized by the direct isomeric product, N-phenyl-N1-methylbenzamimethylation of 2-benzylthio-4(5)-phenylimidazole2 dine was formed in only 0.6% yield. Analowith methyl sulfate and alkali. The odor of gously, N-phenyl-S-benzylisothiourea reacted with mercaptan indicated that some cleavage of the phenacyl bromide to form the imidazole VI in good sulfide was taking place. Orientation in the yield; the isomer obtained corresponds to primary methylation of 2-benzylthio-4 ( 5 )-phenylimidazole alkylation of the nitrogen holding the phenyl was similar to that in the methylation of 4(5)- group with phenacyl bromide. On the other phenylimidazole. Pyman and co-workers3 found hand, N-methylbenzamidine monomethylated in that 4(5)-phenylimidazole, on treatment with Poor Yield and gave a mixture of the two expected isomersS5 Similarly, S-benzyl-N-methyl(1) Abstracted from a thesis by Frank Ross presented t o the reacted Poorly with phenacyl bromide Graduate Faculty of the University of Minnesota, in partial fulfillH-C

‘N-C-H

/I

H-S-C

\N-CH

11

ment of the requirements for the M.S. degree, June, 1949. (2) R. M. Dodson, THISJ O U R N A L , TO, 2753 (1948). (3) Hazeldine, Pyman and Winchester, J . Chcm. Soc., 115, 1431 (1924).

(4)The cleavage of sulfides with phosphorus and iodine in glacial acetic acid may prove t o be a general reaction. This possibility is being investigated. ( 5 ) F. L. Pyman, J . Chcm. SOC.,123, 3359 (1923).